Once established, human tumors are not rejected by the immune system, a state of functional tolerance which eventually proves fatal to the host (Smyth, M. J., et al., Nat. Immunol. 2, 293 (2001)). Evidence from murine models suggests that immunologic unresponsiveness may arise when tumor-associated antigens are presented by certain bone marrow-derived tolerogenic (tolerance-producing) antigen-presenting cells (APCs) (Sotomayor, E. M., et al., Blood, 98: 1070-1077 (2001); Doan, T., et al., Cancer Res., 60: 2810-2815 (2000)). In the setting of tissue transplantation, it would be desirable to isolate and administer such tolerogenic APCs. However, in humans and other mammals (other than mice), the identity of these APCs, and the mechanisms they use to induce tolerance, remain elusive.
In humans, “immature” myeloid dendritic cells (DCs) have been postulated to function as tolerizing APCs based on findings that these cells: (1) have a decreased ability to stimulate T cell responses in vitro (Reddy, A., et al., Blood, 90: 3640-3646 (1997); Jonuleit, H., et al., Eur. J. Immunol., 27: 3135-3142 (1997)); (2) may promote the function of immunosuppressive or “regulatory” T cells following prolonged co-incubation (Jonuleit, H., et al., Trends Immunol., 22: 394-400 (2001)); and (3) have the ability to abrogate antigen-specific T cell responses in vivo (Dhodapkar, M. V., et al., J. Exp. Med., 193: 233-238 (2001); see also U.S. Pat. Nos. 5,871,728 and 6,224,859). However, the molecular mechanism used by immature DCs or other putative tolerogenic APCs to suppress T cell responses is unclear. Moreover, there is currently no way to identify or isolate tolerogenic APCs in vitro or in vivo, and thus, their use as therapeutic agents is still not available for most applications.
More fundamentally, the supposition that immature DCs are tolerogenic is based on an unproven and potentially flawed model of how APCs regulate T cell activation. Thus, a prevailing model teaches that T cells are rendered unresponsive (or “tolerized”) when they receive an activation signal (signal 1) via the T cell antigen receptor (TCR) without receiving co-stimulatory signals (e.g. from CD80 and CD86) delivered on APCs (signal 2). Immature DCs express low levels of TCR ligands (such as MHC class II antigens) and low levels of the putative costimulatory molecules. Thus, the model teaches that immature of DCs are unable to activate T cells because T cells receive signal 1 without adequate signal 2.
Other findings teach against the prevailing model, and indicate that maturation of DCs is not necessarily associated with abrogation of T cell suppression and/or tolerance (Albert, M. L., Nature Immunol., 2: 1010 (2001); Shortman, K. et al., Nature Immunol., 2: 988-989 (2001); T. Bankenstein and T. Schuler, Trends in Immunol., 23: 171-173 (2002)). Instead, there may be a third, as yet undefined signal (signal 3) that acts after T cells have received the signals of antigen presentation and co-stimulation (i.e. signals 1 and 2) from a fully mature APC. The third signal then diverts T cells to activation or tolerance. In this model, the tolerogenic phenotype is independent of the maturation status of the APC (in fact, maturation enhances tolerance induction) and depends instead on an intrinsic attribute of the APC (i.e. whether it expresses signal 3).
The inventors believe that most DC preparations are in fact mixtures of immunizing (stimulatory) and tolerizing APCs. The presence of a mixed population of DCs in such preparations would explain why therapeutic immunization in cancer patients using DCs remains problematic, with most studies having only limited success (M. A. Morse and H. K. Lyerly, Curr. Opin. Mol. Ther., 2: 20 (2000)). For example, the preferred source and differentiation status of DCs for clinical use remains controversial (Curiel T. J., and Curiel, D. T., J. Clin. Invest., 109: 311-312, 2002). Although development of the field has been assisted by the recognition that the maturation state of human DCs plays an important role in their ability to stimulate effective immunity (Dhodapkar, M. V., et al., J. Clin. Invest., 105: R9-R14 (2000); Dhodapkar, M. V., et al., J. Exp. Med., 193: 233-238 (2001)), even using the best isolation and maturation strategies and multiple tumor antigens, clinically useful therapeutic immunization in patients with established tumors has been only partially effective (Banchereau, J., et al., Cancer Res., 61: 6451-6458 (2001)). Thus, it would be useful to develop methods to isolate DCs which, rather than being a mixed population of activating and suppressive DCs, comprise pure activating DCs.
Conversely, these are some situations where increased tolerance to foreign antigens is desired. In one approach, immature dendritic cells (DCs) uncharacterized as suppressive or immunogenic subsets are propagated in the presence of a cytokine regimen to maintain the cells in an immature state. The immature cells are administered to a host in advance of a transplant to enhance tolerance (U.S. Pat. Nos. 5,871,728 and 6,224,859). However, this approach inherently sacrifices efficient antigen presentation and co-stimulation due to the immaturity of the APCs, and risks delivering unwanted immunizing (non-tolerogenic) DCs as part of the heterogeneous DC population. It would be helpful in transplant therapeutics to be able to create well-characterized populations of mature maximally effective tolerogenic APCs which present the antigen subset of interest, but in a tolerizing (tolerance-promoting) preparation.
What is needed is a way to separate tolerance-inducing APCs from other (non-tolerance-inducing) APCs. The tolerance-inducing APCs can then be used in transplant procedures to promote tolerance to specific donor antigens. The non-tolerance-inducing APCs can be used in conjunction with undesirable foreign antigens (such as tumor antigens) as a vaccine, to prime the recipient immune system against the antigen in question.